A cell possesses thousands of unique proteins that serve structural, enzymatic or signaling functions. The intracellular environment is composed of a myriad of structures and membrane-enclosed compartments. The correct subcellular localization is critical for the proper functioning of many proteins. Proteins situated at the lipid bilayer membrane are classified as peripheral proteins, whereas proteins situated within the lipid bilayer membrane are classified as integral membrane proteins. Integral membrane proteins possess intrinsic hydrophobic regions which are inserted into the lipid bilayer as they are synthesized. Typically, peripheral membrane proteins are less tightly associated with membranes and are localized to the lipid bilayer by protein—protein interactions, by intrinsic hydrophobic properties or by the post-translational addition of a lipid group.
A major class of peripheral membrane proteins, known as prenylated proteins, are modified by isoprenoids on a so-called CAaa1Aaa2Xaa (CAAX) motif, wherein C is cysteine, Aaa1 and Aaa2 are aliphatic amino acids and Xaa is any amino acid. This tetra-peptide sequence is located at the proteins' carboxyl termini and triggers a series of modification reactions. Of the approximately 30 known CAAX-containing proteins, the Ras family of small GTP-binding proteins are major constituents. Ras proteins localize at the inner surface of the plasma membrane where they function as key components of various signal transduction pathways or participate in cytoskeletal organization and establishment of cell polarity. The critical role of the Ras proto-oncogene in controlling cell division is exemplified by the participation of mutated forms of the Ras protein in a variety of human tumors, including colorectal carcinoma, exocrine pancreatic carcinoma and myeloid leukemias. Forms of Ras in cancer cells have mutations that distinguish the protein from Ras in normal cells.
The presence of the CAaa1Aaa2Xaa motif sequence targets the protein for at least 3 post-translational modifications. Generally, such modifications include prenylation of the cysteine amino acid, proteolytic removal of the terminal three amino acids (i.e., the Aaa1Aaa2Xaa tripeptide) and methylesterification of the prenylated cysteine, i.e., the C-terminus. More particularly, in the first step, a 15 carbon farnesyl or a 20 carbon geranylgeranyl isoprenyl lipid is added to the cysteine residue. The lipid which is added depends upon the amino acid at the “X” position. Following prenylation, the terminal tripeptide, i.e., the Aaa1Aaa2Xaa tripeptide, is removed by a membrane-bound endoprotease. Thereafter, the resulting C-terminal isoprenylated cysteine is methylesterified.
It has been determined that prenylation of the CAAX motif is essential for the proper functioning of every prenylated protein that has been tested to date. However, the functional requirement of CAAX proteolysis has not been rigorously evaluated because the gene encoding the protease has been elusive. This is true despite the fact that the entire yeast genome has been sequenced and the sequences deposited in GenBank. Unfortunately, elucidation of the complete yeast genome in the absence of functional information for each yeast gene is insufficient for identification of any particular gene. Although many open reading frames (ORFs) have been identified, it is not known whether these ORFs encode functional mRNAs.
Kato, et al. (Proc. Natl. Acad. Sci. USA, 89:9554–9558 (1992)) monitored foci formation of NIH3T3 cells transformed with activated forms of Ras with altered CAAX sequences. They found that one sequence, CVYS, when substituted for the normal Ras CAAX sequence appeared not to have undergone proteolysis and resulted in approximately 50% reduction in foci formation. Unfortunately, the design of this experiment was not ideal because it relied on the heterologous expression of Ras from an SV40 promoter, which resulted in a considerably higher expression level than the physiological Ras promoter. Moreover, the CAAX sequence CVYS displayed a prenylation defect.
In view of the foregoing, there remains a need in the art for the identification of the genes encoding the polypeptides that participate in the post-prenylation modification reactions so that the functional importance of such enzymes can be elucidated.